Diversity and potential plant growth promoting capacity of seed endophytic bacteria of the holoparasite Cistanche phelypaea (Orobanchaceae)

Salt marshes are highly dynamic, biologically diverse ecosystems with a broad range of ecological functions. We investigated the endophytic bacterial community of surface sterilized seeds of the holoparasitic Cistanche phelypaea growing in coastal salt marshes of the Iberian Peninsula in Portugal. C. phelypaea is the only representative of the genus Cistanche that was reported in such habitat. Using high-throughput sequencing methods, 23 bacterial phyla and 263 different OTUs on genus level were found. Bacterial strains belonging to phyla Proteobacteria and Actinobacteriota were dominating. Also some newly classified or undiscovered bacterial phyla, unclassified and unexplored taxonomic groups, symbiotic Archaea groups inhabited the C. phelypaea seeds. γ-Proteobacteria was the most diverse phylogenetic group. Sixty-three bacterial strains belonging to Bacilli, Actinomycetes, α-, γ- and β-Proteobacteria and unclassified bacteria were isolated. We also investigated the in vitro PGP traits and salt tolerance of the isolates. Among the Actinobacteria, Micromonospora spp. showed the most promising endophytes in the seeds. Taken together, the results indicated that the seeds were inhabited by halotolerant bacterial strains that may play a role in mitigating the adverse effects of salt stress on the host plant. In future research, these bacteria should be assessed as potential sources of novel and unique bioactive compounds or as novel bacterial species.

www.nature.com/scientificreports/ The aim of our study was to investigate the diversity of the endophytic bacterial community of surface sterilized seeds of C. phelypaea growing in Iberian coastal salt marshes. We also studied the in vitro PGP traits of the bacterial isolates, such as production of indole, organic acids and siderophores and the ACC deaminase activity.

Results
Seed endophytic bacterial community composition. We used high-throughput sequencing methods to investigate the diversity and composition of the bacterial endophytic community from seeds of C. phelypaea. Alpha diversity indices (Shannon-Wiener biodiversity index, Chao1 and Simpson indexes) were 3.12, 165, 0.868 respectively with P value 0.05. A total of 263 different Operational Taxonomic Unit (OTU)s at genus level were found from 23 phyla (> 0.1%). Unclassified and unexplored taxonomic groups were found as well.
Proteobacteria (48.8%) and Actinobacteriota (43.0%) were the predominating phyla in four replicates. Bacteroidetes (1.2%) and Firmicutes (5.2%) were observed only in two and three out of the four replicates respectively (Fig. 1). Other phyla represented less than 1%. Besides well-known bacterial groups, the seeds were also colonized by newly classified or unknown ones. Among those was the recently proposed bacterial candidate phylum Entotheonellaeota. The other special phyla detected in the C. phelypaea seeds were the newly defined superphylum Patescibacteria 59 , and the phyla Armatimonadetes 60 and Desulfobacteriota proposed by phyl. nov. 61 . The C. phelypaea seeds were also inhabited by symbiotic Archaea belonging to the Nanoarchaeota, Halobacterota and Crenarchaeota.
From the three classes of Proteobacteria, the γ-Proteobacteria were the most diverse compared to α-and β-Proteobacteria. Unclassified Proteobacteria at different taxonomic levels were found as well. Within the α-Proteobacteria, the orders Sphingomonadales and Rhizobiales were found in much higher abundances. At the order level γ-Proteobacteria was dominated by Enterobacterales and Pseudomonadales, and β-Proteobacteria were represented by Burkholderiales and Neisseriales ( Table 1). The phylum Actinobacteriota was mainly represented by the orders Propionibacteriales with Cutibacterium at genus level, and Micromonosporales with the genera Micromonospora and Microbacterium. Besides well-known Actinobacteria, about 8.7% rare Actinobacteria 62 were found: Cryptosporangium, Actinokineospora, Pseudonocardia, Actinoplanes, Kineosporia, and Nocardia. Finally, the third dominating phylum identified in the examined seeds were the Firmicutes with as most important orders the Lactobacillales and the Staphylococcales, with the genera Streptococcus and Staphylococcus respectively. From the order Bacillales, the families Bacillaceae and Planococcaceae were represented. More detailed information about dominating endophytic bacteria at different taxonomic levels is presented in Table 1.
Diversity and in vitro characterization of PGP bacteria isolated from the surface-sterile seeds. In order to obtain as complete as possible overview of the culturable members of the endophytic community of C. phelypaea seeds, different culture media were chosen. In total 63 bacterial strains belonging to Firmicutes (66.7%), Actinobacteriota (15.9%), α-, γ-and β-Proteobacteria (14.3%) and unclassified bacteria (3.2%) were picked up from the 1/869 rich, Flour1, PDA, R2A, 284, and YEDC media and subsequently identified ( Table 2). The morphological characteristics of bacterial colonies and more detailed taxonomic information about isolated bacterial strains were presented in Supplementary Table S1 and Supplementary Fig. S1.
The isolated strains were also tested in vitro for various Plant Growth Promoting (PGP) properties which are presented in Fig. 2. Respectively, 41 (65.1%) and 37 (58.7%) of all isolated strains tested positive for the production of indole (IAA) and organic acids (OA) (Fig. 2a); among those the strains belonging to the genera  www.nature.com/scientificreports/ Ralstonia, Paenibacillus, Peribacillus demonstrated the highest IAA production (Fig. 2b). Meanwhile, the strains belonging to Micromonospora, Peribacillus and unclassified strains demonstrated more positive responses for the production of organic acids (Fig. 2b). For both, ACC-deaminase and siderophore production, respectively 20 (31.7%) and 21 (33.3%) of the investigated strains tested positive (Fig. 2a); among those 75% of the Micromonospora and 100% of the Ralstonia were positive for production of siderophores. In case of ACC deaminase, most positive responses were found for strains belonging to Micromonospora, Ralstonia and Paenibacillus (Fig. 2b). Among the β-Proteobacteria, Ralstonia spp. demonstrated the highest number of positive responses for siderophore production. Among the Actinobacteria, Micromonospora spp. demonstrated equally high positive responses for all tested PGP traits (Fig. 2b). Detailed information about the in vitro PGP traits is presented in Supplementary Table S1 and Supplementary Fig. S1.
Salt tolerance of cultivated bacteria. In order to confirm the salt tolerance of the isolated strains the modified Brain-heart infusion (BHI) broth 63 was used. After 7-10 days observation all tested bacterial strains demonstrated salt tolerance ( Supplementary Fig. S1). Table 1. Cumulative list of dominating endophytic bacteria in the seeds of Cistanche phelypaea and their taxonomic information. Only taxonomic groups with an abundance > 10% are listed. *Other genera were represented at abundances lower than 10% of the total seed endophytic community.  www.nature.com/scientificreports/

Discussion
Salt marshes are natural habitats for many unique salt-tolerant plants 64 . Among these, the holoparasitic C. phelypaea can be highlighted. The heterotrophic lifestyle of parasitic plants was leading to several morphological, physiological and molecular adaptations with unique properties that make them an exciting group for study; the production of large numbers of very small seeds and the very specific conditions of germination are examples. Besides, they have significant effects on the ecosystems in which they occur 15 . The importance of wetlands, particularly salt marshes and the limited knowledge concerning the microbiology of such systems form the  www.nature.com/scientificreports/ incentive for the current paper, focusing on the seed-associated endophytic bacterial community of the obligate root parasite C. phelypaea that is parasitizing on the roots of halophytic plant species 6 . In a recent study 32 we examined the bacterial seed endomicrobiome of another species from the genus Cistanche, C. armena, an endemic species growing in saline and arid habitats in Armenia. The seed endophytic bacterial community of C. armena is less diverse compared to that of C. phelypaea and consists mainly of spore forming, halophytic bacteria, with the Firmicutes as the predominating phylum (60.4%) ( Table 3). From the seeds of C. armena, 10 bacterial phyla and 256 genera were identified. The structure and diversity of this endophytic bacterial community is related to the natural habitat of their host plant 32 .
In the present study we describe the structure and diversity of the endophytic bacterial community of seeds from an endemic C. phelypaea population growing in coastal salt marshes in Portugal. Proteobacteria, Actinobacteriota, Firmicutes and Bacteroidetes are well represented (> 1%) in surface sterile seeds (Fig. 1). Corresponding results were reported for the seeds of other holoparasitic Orobanchaceae species, C. armena 32 and Phelypanche ramosa 54 .
Besides commonly known bacterial groups, the seeds C. phelypaea are also colonized by newly classified or unexplored bacterial phyla that have not been reported in such seeds before. Among these are Entotheonellaeota which, until now, were found mainly associated with marine sponges. Bacteria isolated from the marine sponge Theonella swinhoei Y are uncultured filamentous bacteria "Candidatus Entotheonella factor" and "Candidatus Entotheonella gemina". Whole genome sequencing of Ca. Entotheonella demonstrated an extraordinarily rich genomic potential for the production of bioactive natural products with unique structures, uncommon biosynthetic enzymology and largely unknown properties 65 . Another interesting newly defined superphylum Patescibacteria has been found in groundwater, sediments, lakes, and other aquatic environments and was found associated with plants as well 59,66 . The role of Patescibacteria in plants is still unclear, although they have been discovered inside of tissues of different plants, in plant associated soils and in degrading plant biomass 60,66 . The phylum Armatimonadetes is assumed to harbor strains that are widely involved in degradation of plant material, substances based on polysaccharides 60 .
In seeds of C. phelypaea, among the Proteobacteria, the γ-Proteobacteria were more diverse compared to αand β-Proteobacteria (Table 1). In comparison, in C. armena seeds were only abundance by γ-Proteobacteria with as dominating orders Xanthomonadales, Pseudomonadales and Enterobacterales 32 . The dominating γ-Proteobacteria in seeds of C. phelypaea were Pseudomonadales and Enterobacterales. At family level, Morganellaceae, Aeromonadaceae, Shewanellaceae, Pseudomonadaceae, Moraxellaceae were the most abundant ( Table 1). Classes of Proteobacteria showed diverse distribution tendencies in different ecosystems. In sediments of marine wetland ecosystems, a wide distribution of γ-Proteobacteria has been reported, and most of them seemed involved in sulfur reduction 67 . However, a high abundance of α-and β-Proteobacteria appeared in freshwater sediments; this should be correlated with pH and nutrients 68 .
The second most dominating bacterial phylum in seeds of C. phelypaea was the Actinobacteriota, with a more or less similar abundance as the Proteobacteria (43.0% against 48.8%). This is different from the seeds of C. armena in which Actinobacteriota represented only 6.5% (Table 3). The abundance of this phylum in the examined seeds is not unexpected, taking into account the habitat in which C. phelypaea is growing. Actinobacteriota indeed were reported more often as the most characteristic phylum in wetland ecosystems 1,3,45,47,48 . Until today, the community structure, diversity, biological activities and mechanisms of environmental adaptation of Actinobacteria to special and extreme environments have been poorly studied compared to more moderate and terrestrial environments 69 . In seeds of C. phelypaea, the most abundant genus of the Actinobacteriota was Micromonospora (38.1%). The genera Microbacterium and Curtobacterium also were dominating genera in seeds of C. armena growing in a saline, extremely arid environment in Armenia 32 . It seems that Curtobacterium has an overall distribution and is present in both, wet and arid ecosystems. Many studies mentioned Curtobacterium being a pathogen of economically important plants 70 . Moreover, this genus is known to harbor a highly diverse genomic potential for the degradation of carbohydrates, particularly with regard to structural polysaccharides 71 .
The seeds of C. phelypaea are colonized by rare Actinobacteria as well: Actinoplanes, Actinokineospora, Cryptosporangium, Kineosporia, Nocardia, Pseudonocardia. These bacteria are relatively difficult to isolate and cultivate in vitro due to difficulties in mimicking the conditions of their natural habitats. Rare Actinobacteria were isolated from different environments: the deep ocean, deserts, mangroves, plants, caves, volcanic rocks, and stones, which illustrates their wide distribution in the biosphere 72 . As reported by several authors, such bacteria are a potential source of novel bioactive compounds. Thus, it is essential to enhance our knowledge about their diversity and distribution in the environment 46,60 .
In case of C. armena seeds, Firmicutes was the predominating phylum with a total abundance of 60.5% ( Table 3). The isolated Firmicutes belonged to the genera Paenibacillus (28%), Bacillus (41.9%) and some other genera (11.8%) 32 . Using a range of different growth media allowed us to isolate and cultivate a quite high diversity Table 3. Comparison of dominant bacterial phyla (> 1%) in seeds of Cistanche armena and C. phelypaea. The predominating phyla are indicated in bold.
Based on in vitro tests, the selected bacterial isolates seem to possess several plant growth-promoting traits. 58.7% of the isolated strains belonging to various genera produced organic acids (OA), and also 65.1% showed able to produce IAA (Fig. 2a). Among those bacterial strains belonging to genera Ralstonia and Peribacillus showed the highest number of positive responses in the in vitro IAA production. In case of organic acids, the highest numbers of positive responses were found among Micromonospora, Peribacillus and Unclassified bacteria (Fig. 2b). Production of IAA and organic acids by many of the seed endophytes originating from coastal salt marshes (30-35% salt concentration) could be expected, since the seed endophytes isolated from C. armena from arid and saline habitats, also demonstrated high levels of IAA and organic acids production 32 . In the present study, 65.1% and 58.7% of isolates produce IAA and organic acids, respectively. Meanwhile, Petrosyan et al. 32 demonstrated that most of bacterial strains isolated from the surface sterilized seeds of C.armena also could produce IAA (80.7%) and organic acids 51.6% (Fig. 3). Numerous studies have demonstrated that IAA-producing halotolerant bacteria increase the tolerance of plants to saline conditions. Organic acids production by rhizosphere microorganisms is one of the mechanisms to solubilize phosphorus (P) included in insoluble mineral compounds in soils 76 . Altogether, the seeds of both Cistanche species are inhabited by halotolerant bacteria that may have a role in mitigating the adverse effects of salt stress in the holoparasitic plant. However, it is still not clear if the PGP traits of these bacteria are beneficial only for the holoparasite either also for its host plant. It also remains unclear if the PGP capacities of seed endophytes may protect the embryo of the holoparasite against salt stress.
ACC deaminase capacity is another important trait of PGP endophytes. Many halophytic plants (Salicornia europaea, Halimione portulacoides, Prosopis strombulifera and Limonium sinense) are colonized by endophytes with ACC deaminase capacity 77 . ACC-deaminase can lower ethylene production under salt stress. Indeed, endophytes with ACC-deaminase capacity convert ACC (1-aminocyclopropane-1-carboxylic acid, the immediate precursor of ethylene in plants) into a-ketobutyrate and ammonia which helps the plants to reduce the negative effects of salinity stress on root development 77 . The major consequences of saline stress are nutrient imbalances and negative effects on plant physiology in general. Siderophores are high affinity iron-chelating compounds. Iron is a micronutrient, essential for plant growth and development, since it is an essential co-factor for a variety of reactions in plant cell metabolism and Fe-requiring enzymes. It is part of several metalloenzymes involved in photosynthesis and respiration. In saline soils, the bioavailability of Fe to plants is low. The role of microbial siderophores in Fe supply to plants and on the mitigation of saline stress in crop growth is well documented 78 . Respectively 33.3% and 31.7% of the isolated bacterial strains tested positive for siderophore and ACC deaminase production (Fig. 2a). Compared to C. armena that 16.1% were positive for siderophore and 42% scoped to positive for ACC deaminase (Fig. 3). Most positive responses were observed for Actinomycetia and β-Proteobacteria. It seems that β-Proteobacteria with Ralstonia spp. have a high siderophore producing capacity (Fig. 2b). Sphingomonas spp. (α-Proteobacteria) demonstrated the lowest level of positive responses for all in vitro tested PGP traits. Quite similar outcomes were observed for production of indole and organic acids. Also similar numbers of endophytes scored positive for ACC-deaminase and siderophore production (Fig. 2a). Among the Actinobacteria, Micromonospora spp. demonstrated the most positive responses for the in vitro tested PGP traits and thus www.nature.com/scientificreports/ might be considered as potentially the most favorable endophytes in C. phelypaea seeds. Janibacter strains, also belonging to the Micrococcales scored negative for all tested PGP assays (Supplementary Table S1).

Conclusion
The holoparasitic C. phelypaea, which is parasitizing halophytic host plants, is the only representative of the genus Cistanche that is found in coastal salt marshes in Portugal. We explored the seed endophytic bacterial community of this holoparasite. We succeeded to isolate 63 bacterial strains, whilst using Illumina paired-end sequencing 263 different bacterial genera from 23 bacterial phyla were identified as seed endophytes of C. phelypaea. The 63 cultivable strains appeared to be salt tolerant and to possess plant growth-promoting traits like production of organic acids, IAA, siderophores and ACC deaminase. The genus Micromonospora harbored the most beneficial bacteria. These halotolerant bacteria have potential to mitigate the adverse effects of salt stress in the holoparasite. Possibly, they may also protect the embryo of the holoparasite against salt stress and maintain the seeds a long time viable. However, to confirm this, further studies concerning the beneficial effects of PGP seed endophytes on the holoparasite are required. In addition, the seeds of C. phelypaea represent and underexplored reservoir of diverse and novel endophytic Actinobacteria that may be of potential interest in the discovery of new bioactive compounds.

Materials and methods
Site description and seed sampling.  (Fig. 4). The Iberian Peninsula is typified as a hotspot area of biodiversity with a high level of endemism 80 . The Ria de Alvor is actually a priority conservation area, is part of the European Ecological Network, Natura 2000 and is a RAMSAR wetland of international importance since 1996 64 . Mature and dry seeds of C. phelypaea were collected from ripe fruits from at least 10 individual plants of the total population from the mentioned location in April 2012. Species were collected and identified by Renata Piwowarczyk. The herbarium materials were deposited in the Herbarium of the Jan Kochanowski University in Kielce (KTC), Poland. Vaucher specimens in the Herbarium KTC are not numbered yet. The herbarium materials are stored in a stable temperature of about 20 °C and a relative humidity of 30%. Field studies, the collection of plant and seeds material was complied with relevant local, institutional, national, and international guidelines, permissions, or legislation, and necessary permissions were obtained.
Seed surface sterilization and homogenization. The aim of seed surface sterilization is to obtain only endophytic bacterial communities (culturable and unculturable). The surfaces of Cistanche seeds possess an alveolate ornamentation sculpture with polygonal and isodiametric cells with different sizes 81 . An efficient sterilization protocol is crucial. For this purpose, the seeds surface sterilization techniques described by Petrosyan et al. 32 were used. The efficiency of the sterilization procedure was verified by plating 100 µl of the last rinsing www.nature.com/scientificreports/ water on 869 rich medium 82 . Subsequently, the surface sterilized seeds were mechanically homogenized in 0.5 ml 10 mM MgSO 4 solution using a sterile pellet pestle (Kimble®). The homogenization process was accomplished using 5-6 metal stainless steel bead balls (2.8 mm) and a two-bladed mixer mill (Retsch MM400, Germany) for 15 min at 25 Hz. Part of the homogenous suspension was stored at − 80 °C for DNA extraction, another part was used for isolation of culturable bacteria (see below).

Amplification of the 16S rRNA gene fragment and preparation of the next-generation sequencing library.
For isolation and identification of the total (cultivable and uncultivable) bacterial community, the homogenized suspension of the surface sterilized seeds was used. The DNA isolation was performed in 4 replicates using the Mobio PowerPlant protocol based on the PowerPlant® Pro DNA Isolation Kit and patented solution (Inhibitor Removal Technology® IRT) for removal of PCR inhibitors from plant extracts during the isolation process. All isolated DNA samples were subjected to bacterial 16S rRNA gene amplicon PCR. In the first round of 16S rRNA gene PCR, an amplicon of 291 bp was generated, using primers 515F-GTG YCA GCMGCC GCG GTAA and 806R-GGA CTA CNVGGG TWT CTAAT 83  Bioinformatic processing of reads. Sequences were demultiplexed using the Illumina Miseq software, and subsequently quality trimmed and primers removed using DADA2 1.10.1 85 in R version 3.5.1. Parameters for length trimming were set to keep the first 290 bases of the forward read and 200 bases of the reverse read, maxN = 0, MaxEE = (2,5) and PhiX removal. Error rates were inferred, and the filtered reads were dereplicated and denoised using the DADA2 default parameters. After merging paired reads and removal of chimeras via the RemoveBimeraDenovo function, an amplicon sequence variant (ASV) table was built and taxonomy assigned using the SILVA v138 training set 86,87 . The resulting ASVs and taxonomy tables were combined with the metadata file into a phyloseq object (Phyloseq, version 1.26.1) 88 . Contaminants were removed from the dataset using the package Decontam (version 1.2.1) applying the prevalence method with a 0.5 threshold value 89 . A phylogenetic tree was constructed using a DECIPHER/Phangorn pipeline as described before 90 .

Data visualization and statistical analyses.
The ASV table was further processed removing organelles (chloroplast, mitochondria), and prevalence filtered using a 2% inclusion threshold (unsupervised filtering) as described by Callahan 85 . Alpha-diversity metrics such as Chao1, Simpson's and Shannon's diversity indexes were calculated on unfiltered data using scripts from the MicrobiomeSeq package. Hypothesis testing was done using analysis of variance (ANOVA) and the Tukey Honest Significant Differences method (Tukey HSD). When assumptions of normality and homoscedasticity were not met, a Kruskal-Wallis Rank Sum test and a Wilcoxon Rank Sum test was performed. The results were summarized in boxplots and relative abundances were calculated and visualized in bar charts using Phyloseq. All performed statistical tests were corrected for multiple testing and alpha < 0.05 was considered as statistically significant. All graphs and figures were generated in R version 4.0.4, Microsoft Office Excel 2010 software.
Isolation and cultivation conditions of culturable endophytes. The first part of the obtained suspension (see above) was used for DNA extraction, the second part was for isolation of culturable bacteria. In order to cultivate as many as possible of the seed endophytes, 100 µL of the seed suspension was plated onto 1/869 rich 82 , Potato Dextrose Agar (PDA) (Sigma-Aldrich), R2A (Sigma-Aldrich), 284 91 , Flour-yeast extractsucrose-casein hydrolysate agar (Flour1) and extract-casein hydrolysate agar (YECD) 92 . Besides the undiluted macerate, serial dilutions were prepared from 10 -1 to 10 -3 cfu ml 1  Genomic DNA extraction and taxonomic identification of the culturable endophytic bacterial strains. The DNA isolation was performed using standard procedure for DNA isolation from bacterial pellets with MagMAX Express 96 (APPLIED BIOSYSTEMS, Finland). DNA was quantified in a Qubit® 2.0 Fluorometer (Thermo Scientific, US) and a Nanodrop spectrophotometer (Thermo Scientific, US) with an A260/A280 ratio of 1.7-2.0 was used for checking the purity. The near full-length sequences of the 16S rRNA gene were amplified using 27f (5-AGA GTT TGATCMTGG CTC AG-3) and 1492r (5-GGT TAC CTT GTT ACG ACT T-3) primers. A reaction volume of 25 μl per sample was containing 1 μl of DNA, 1 × Q5 Reaction Buffer (2 mM MgCl 2 ), 0.25 μM 27f and 1492r primers, 200 μM dNTP mix and 0.02 U µl −1 Q5 High-Fidelity DNA polymerase and 1 × 5 × Q5 High GC Enhancer 84 . The 1/10 and 1/20 dilution for some DNA samples were done. PCR conditions were the same as described above, but the number of cycles reduced to 30, and 55 °C annealing temperature. The samples for 16S rRNA Sanger sequencing were shipped to Macrogen (https:// www. macro gen-europe. com/). Sequences were quality filtered using Geneious v4.8 and were analyzed using the ribosomal database SILVA (https:// www. arb-silva. de/ align er/). Bacteria were identified based on the PCR amplification of partial 16S rDNA gene sequences and annotated using the NCBI GenBank databases by the program Standard Nucleotide BLAST and RDP database (https:// rdp. cme. msu. edu/ seqma tch/ seqma tch_ intro. jsp).

Plant growth promoting (PGP) traits.
Considering the growth conditions of C. phelypaea the endophytic bacterial isolates were tested for their in vitro plant growth promoting traits and salt tolerance in high salt concentration (6.5%). All tests were performed at least two times.
The tryptophanase activity and Indole-3-acetic acid (IAA) production ability was tested using the Salkowski reagent 32 . The organic acid production was determined using the method of Cunningham and Kuiack 93 . ACCdeaminase activity was tested in SMN medium with 5 mM ACC 94 . Production of siderophores was assessed by using the 284 medium with 0.25 μl optimal iron concentration with CAS solution 95 . The detailed descriptions of methods were presented previously 32 .
Salt tolerance. Salt tolerance ability of isolated bacteria was tested using the modified Brain-heart infusion (BHI) broth 63 with composition: Peptic digest of animal tissue 10 g; Heart infusion 10 g; Glucose 1 g; Sodium chloride 65 g; Bromocresol purple 0,016 g per liter with pH 7.2. Bacteria were incubated for 5-7 days at 30 °C at 150 rpm agitation. After 7 days reaction time, the samples were observed for visible growth (turbidity) or color change. The bacterial growth (increase in turbidity) and/or change in the color of broth from purple to yellow was considered as positive. BHI without bacteria was chosen as negative control.
Taxonomic classification of isolates and delineation of molecular OTUs. Ribosomal RNA gene sequences from bacterial isolates were compared with reference sequences from the GenBank databases, using Basic Local Alignment Search Tool (BLAST) software (http:// blast. ncbi. nlm. nih. gov/ Blast. cgi) and the Ribosomal Database Project (RDP) website (http:// rdp. cme. msu. edu/), and isolates were assigned to species or the highest taxonomic rank possible.

Data availability
The sequence data available in the NCBI Genbank (https:// www. ncbi. nlm. nih. gov/) Sequence Read Archive with BioSample accession number SAMN26931786. The partial 16S rRNA gene sequences received from of all studied strains were deposited in the GenBank database under the accession numbers presented in Supplementary Table S1. www.nature.com/scientificreports/